Scientists have designed a new molecular tool, dubbed
"LigAmp," to pinpoint DNA mutations among thousands of
cells, the equivalent of searching for a single typo in an
entire library of books. Preliminary studies in a small
number of cell lines and body fluids show the
ultra-sensitive test may help detect microscopic cancer and
HIV drug resistance.
"Other molecular tests make it very difficult to
locate a mutation in a particular cell surrounded by
thousands of other cells that don't have the mutation,"
says James Eshleman, who led the study with colleagues from
the Johns Hopkins
Department of Pathology and
Kimmel
Cancer Center. "LigAmp essentially filters background
'noise' caused by normal cells and reveals specific
mutations."
The researchers say that sensitive tests to locate
mutations could identify cancer in patients at high risk
for the disease. Such tests could even help detect a
recurrence of cancer by monitoring whether the number of
mutations rises above a predetermined threshold value.
In addition to cancer detection, the Johns Hopkins
mutation-finder appears able to detect drug-resistant HIV.
The team tested it on blood samples from a handful of
patients with HIV and located DNA mistakes in the virus
itself that make it resistant to certain antiretroviral
drugs. Results of analyses of the new test are published in
the November issue of Nature Methods.
"We designed LigAmp to improve how we look for
extremely subtle variations in viral and cellular DNA,"
says Eshleman, an associate professor of pathology and
oncology in the School of Medicine and associate director
for the DNA Diagnostics Laboratory at Johns Hopkins. "The
molecular code of normal cells may look identical to
cancerous except for a single rung in the DNA
ladder-structure."
The test works by creating a molecular "magnet" with
an affinity for the DNA mistake, also known as a point
mutation. If the mutation is found, the magnet binds to it
and inserts a bacterial gene. The bacterial gene serves as
a red flag and produces a fluorescent color visible to
powerful computer programs.
In their studies, the Hopkins investigators tested
LigAmp on colon cancer cell lines, blood from HIV patients
and fluid from cancer patients' pancreatic ducts. Single
mutations in colon cancer cells and drug-resistant HIV
viruses were detected at dilutions of up to one in 10,000
molecules. Mutations of the KRAS2 gene were detected in
duct fluid samples from three pancreatic cancer patients,
which also corresponded to mutations found in their tumors.
LigAmp also located a drug-resistant mutation, called
K103N, in blood samples from three HIV patients.
Further analysis of LigAmp with larger sample sizes
and blinded panels of clinical samples currently is under
way.
"Some initial studies show that we can simultaneously
look for different mutations and quantify the number of
mutated molecules present. This may help us build panels of
cancer markers for screening and determine low or high
levels of mutation," Eshleman said.
Funding for this research was provided by the Maryland
Cigarette Restitution Fund, the National Cancer Institute
and the National Institute of Allergy and Infectious
Diseases.
Johns Hopkins colleagues working with Eshleman are
Chanjuan Shi, Susan Eshleman, Dana Jones, Noriyoshi
Fukushima, Li Hua, Antony Parker, Charles Yeo, Ralph Hruban
and Michael Goggins.
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